Characterization of an Omnitrap-Orbitrap Platform Equipped with Infrared Multiphoton Dissociation, Ultraviolet Photodissociation, and Electron Capture Dissociation for the Analysis of Peptides and Proteins

We describe an instrument configuration based on the Orbitrap Exploris 480 mass spectrometer that has been coupled to an Omnitrap platform. The Omnitrap possesses three distinct ion-activation regions that can be used to perform resonant-based collision-induced dissociation, several forms of electron-associated fragmentation, and ultraviolet photodissociation. Each section can also be combined with infrared multiphoton dissociation. In this work, we demonstrate all these modes of operation in a range of peptides and proteins. The results show that this instrument configuration produces similar data to previous implementations of each activation technique and at similar efficiency levels. We demonstrate that this unique instrument configuration is extremely versatile for the investigation of polypeptides.


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(A) Schematic drawing of the Orbitrap-Omnitrap instrument S3 Figure S1(B) Schematic drawing of the cross-section of the Omnitrap and IR laser beam propagating along the axis S4 Figure S2 Schematic drawing of segment Q5 and overlap between an ion cloud and an IR laser beam in Q5 S5 Figure S3 Schematic drawing of segments Q7,Q8, Q9 and overlap between an ion cloud and a UV laser beam in Q8 S6 Figure S4 Pressure profile in the Omnitrap following a pulse of buffer gas S7 Figure S5 Dependence of IRMPD efficiency on the delay between a gas pulse and IR triggering S8 Figure S6 Dependence of IRMPD efficiency on the q value S9 Figure S7 Dependence of IRMPD efficiency on the length of IR pulse S10 Figure S8 IRMPD spectra of [Glu-fibrinopeptide B] 2+ acquired in segments Q2, Q5 and Q8 S11 Figure S9(A,B,C) IRMPD spectra of ubiquitin 8+ , myoglobin 10+ and [carbonic anhydrase] 20+ acquired in segment Q5 of the Omnitrap and corresponding fragment mapping S12 Figure S9(D,E) Effect of IR laser power on the abundance of internal fragments in IRMPD S13 Figure S10 Normalised intensities of few selected fragments in the UVPD of [bradykinin] 2+ for different number of pulses of the UV laser S14 Figure S11 Normalised TIC of all main-series fragments and normalised intensity of unfragmented precursor in the UVPD of ubiquitin 8+ for different number of pulses of the UV laser S15 Figure S12 UVPD spectra of ubiquitin 8+ and [carbonic anhydrase] 20+ S16 Figure S13 Number of fragments of different types identified in the UVPD experiments of ubiquitin 8+ S17 Figure S14 Fragment maps for UVPD experiments on ubiquitin 8+ , myoglobin 10+ , and [carbonic anhydrase] 20+ S18 Figure S15 UVPD and IR-activated-UVPD spectra of myoglobin 10+ S19 Figure S16 Ion currents of fragments of different types and sequence coverages identified in the IR-activated-UVPD experiments of myoglobin 10+ S20 Figure S17 UVPD and IR-activated-UVPD spectra of [carbonic anhydrase] 20+ S21 Figure S18 Ion currents of fragments of different types and sequence coverages identified in the IR-activated-UVPD experiments of [carbonic anhydrase] 20+ S22 Figure S19 ECD spectra of ubiquitin 8+ , myoglobin 20+ , and [carbonic anhydrase] 20+ S23 Figure S20 Fragment maps of ECD of ubiquitin 8+ , myoglobin 20+ , and[carbonic anhydrase] 20+ S24 Figure S21 Fragment maps of ECD and IR-activated-ECD of myoglobin 10+ S25 Figure S22 Sequence coverages by fragments of different types identified in the ECD and IR-activated-ECD experiments of myoglobin 10+ S26 Figure S23 EID and IR-activated-EID spectra of myoglobin 10+ S27 Figure S24 Fragment maps of EID and IR-activated-EID of myoglobin 10+ S28 Figure S25 Sequence coverages by fragments of different types identified in the EID and IR-activated-EID experiments of myoglobin 10+ S29 Figure S26 ECD and IR-activated-ECD spectra of [Glu-fibrinopeptide B] 2+ S30 Figure S27 Distributions of normalised intensities of all c and z fragments identified in IR-activated-ECD of [Glu-fibrinopeptide B] 2+ for different power outputs of the IR laser S31 Figure S28 Summed ion currents of a,b,c,y,z fragments identified in IR-activated-ECD of triply charged chain B of insulin for different power outputs of the IR laser S32  Table S1 Details about analytes used in the experiments S33 Table S2 Types of fragments searched in the spectra using the in-house software and MS-TAFI S34  Figure S1. A) Schematic drawing of the Orbitrap-Omnitrap instrument. B) Schematic drawing of the cross-section of the Omnitrap in the plane parallel to its axis and the overlap between an ion cloud (500000 charges) trapped at q=0.15 in Q2/Q5/Q8 and an IR laser beam. The radial density of the ion cloud is represented by a normal distribution with a variance (σ) of ~0.4 mm. The variation corresponding to 4σ covers approximately 99% of the population of charges. The ideal case of a laser beam perfectly aligned along the axis is considered.

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beam path tubing mirror focal lens

mm
ion cloud (4σ r = ~1.6 mm) B) Figure S1. A) Schematic drawing of the Orbitrap-Omnitrap instrument. B) Schematic drawing of the cross-section of the Omnitrap in the plane parallel to its axis and the overlap between an ion cloud (500000 charges) trapped at q=0.15 in Q2/Q5/Q8 and an IR laser beam. The radial density of the ion cloud is represented by a normal distribution with a variance (σ) of ~0.4 mm. The variation corresponding to 4σ covers approximately 99% of the population of charges. The ideal case of a laser beam perfectly aligned along the axis is considered.

S4
IR beam (d = ~1 mm) inscribed radius (r = 4 mm) Figure S2. Schematic drawing of the cross-section of the segment Q5 in the plane orthogonal to the axis of the trap and the overlap between an ion cloud (500000 charges) trapped at q=0.15 and an IR laser beam. The radial density of the ion cloud is represented by a normal distribution with a variance (σ) of ~0.4 mm. The variation corresponding to 4σ covers approximately 99% of the population of charges.

S5
UV beam (~10 μm) 8 mm 12 mm ion cloud (L = ~16 mm) Figure S3. Schematic drawing of the cross-section of the segments Q7, Q8, Q9 in the plane parallel to the axis of the trap and the overlap between an ion cloud (500000 charges) trapped at q=0.15 and a UV laser beam. The radial density of the ion cloud is represented by a normal distribution with a variance (σ) of ~0.4 mm. The variation corresponding to 4σ covers approximately 99% of the population of charges.